Highly reproducible and cost-effective one-pot organoid differentiation using a novel platform based on PF-127 triggered spheroid assembly.

Organoid technology offers sophisticated in vitro human models for basic research and drug development. However, low batch-to-batch reproducibility and high cost due to laborious procedures and materials prevent organoid culture standardization for automation and high-throughput applications. Here, using a novel platform based on the findings that Pluronic F-127 (PF-127) could trigger highly uniform spheroid assembly through a mechanism different from plate coating, we develop a one-pot organoid differentiation strategy. Using our strategy, we successfully generate cortical, nephron, hepatic, and lung organoids with improved reproducibility compared to previous methods while reducing the original costs by 80-95%. In addition, we adapt our platform to microfluidic chips allowing automated culture. We showcase that our platform can be applied to tissue-specific screening, such as drug toxicity and transfection reagents testing. Finally, we generate NEAT1 knockout tissue-specific organoids and show NEAT1 modulates multiple signaling pathways fine-tuning the differentiation of nephron and hepatic organoids and suppresses immune responses in cortical organoids. In summary, our strategy provides a powerful platform for advancing organoid research and studying human development and diseases.